Technical Abstract: A series of experiments were conducted to evaluate the effects of ergot alkaloids (dihydroergotamine, ergonovine, and ergotamine) on E. coli O157:H7 in both pure and mixed ruminal fluid culture. Alkaloids were added to solutions of E. coli O157:H7 strains 933 (pure and ruminal cultures) and 6058 (ruminal culture only), and growth rates and colony forming units (CFU) of E. coli O157:H7 were measured. A combination of all three alkaloids at either 2 or 500 µM decreased (P < 0.001) the growth rate of E. coli O157:H7 in pure culture compared to the individual alkaloids. Dihydroergotamine tended (P = 0.07) to reduce growth rate of E. coli O157:H7 in pure culture compared with ergonovine or ergotamine alone. Increased concentrations of dihydroergotamine and ergotamine decreased (P < 0.003) growth rate of E. coli O157:H7 but increasing concentrations of ergonovine did not influence (P > 0.10) E. coli O157:H7 growth rate. Similar to results in pure culture, a combination of all three alkaloids, independent of concentration, decreased (P < 0.001) the CFUs of E. coli O157:H7 strain 6058 in mixed ruminal culture compared to the individual ergot alkaloids. Dihydroergotamine decreased (P = 0.04) CFUs of E. coli O157:H7 strain 6058 when compared to ergonovine but CFUs was similar (P > 0.10) between dihydroergotamine and ergotamine. Ergot alkaloids commonly found in endophyte-infected tall fescue plants reduced E. coli O157:H7 in vitro. Ruminal and(or) intestinal populations of E. coli O157:H7 may be influenced in livestock consuming endophyte-infected tall fescue, and these alterations could be due to the presence of ergot alkaloids in fescue plants.